【作者】 徐莹；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品科学， 2005， 博士

【摘要】 弹性蛋白酶是一种以水解不溶性弹性硬蛋白为特征的广谱蛋白水解酶。可以由动物胰脏提取,也可以由微生物发酵法生产。弹性蛋白酶可以作为治疗高血脂症、防治动脉粥样硬化症等的生化药物;在食品工业中主要作为肉类嫩化剂,此外在动物蛋白水解、高级保健化妆品等方面均有应用。 本文以本实验室保藏的高产弹性蛋白酶菌株Bacillus sp.EL31410及其发酵的弹性蛋白酶为研究对象,采用优化的发酵培养基进行了5L发酵罐分批发酵工艺条件优化,并利用70L发酵罐完成了中试试验,从发酵液中纯化出弹性蛋白酶并研究其酶学性质,筛选并优化出提高弹性蛋白酶热稳定性的安全复合保护剂,估算了弹性蛋白酶的贮存寿命,对双水相体系纯化弹性蛋白酶也进行了研究,还探讨了菌源性弹性蛋白酶水解丝素蛋白的能力。主要结论如下: 1) 在5L发酵罐内应用好氧微生物培养技术进行分批发酵,研究其培养过程的特性,进行相应地动力学分析,建立了最适培养策略。温度对弹性蛋白酶分批发酵影响是明显的。37℃培养很适合该芽孢杆菌早期生长,当置于30℃培养时弹性蛋白酶酶活力最大。通过比较四种不同的温度培养模式后发现,30℃培养非常适合Bacillus sp.EL31410生产弹性蛋白酶。通过比较不同恒定pH进行Bacillus sp.EL31410分批发酵过程发现,pH对细胞的生长和弹性蛋白酶的合成影响很大。在恒pH 6.5和7.0时,细菌的生长情况都较好。恒定pH 7.0获得弹性蛋白酶活力值最高。提出了分段控制恒定pH策略,即0到24h恒定pH6.5,24h后恒定pH7.0,经验证,最大酶活力为699U/mL,48h达到产酶高峰。采用分段控制恒定pH策略,保持通气量不变,将搅拌转速由300rpm提高到400rpm,泡沫仍然能够控制,细胞的生长和产酶情况良好,并且在保持最大酶活力相当的情况下,发酵周期提前6h。 2) 根据5L发酵罐分批发酵的发酵工艺参数进行合理放大,调整后的70L发酵罐分批发酵参数为:培养温度30℃,通气量为1vvm,搅拌转速为230rpm,发酵过程中采用分段控制pH策略。发酵结果与5L罐发酵结果接近,在培养33h进入产酶高峰期,酶活力达到645U/ml。实现了成功地放大,为工业化生产奠定了基础。用Logistic方程、更多还原

【Abstract】 Elastase is a kind of protease that can specially degrade elastin. Elastase comes from animal pancreas and also from microbe culture. Elastase production by fermentation was more promising when its activity beyond 155 U/mL. For its special enzymatic characteristics, it has broad applications in medical therapy, in food processing, in functional cosmetics and so on.In this study, using high yield elastase-producing strain Bacillus sp. EL 31410 which preserved by our lab and its product—elastase, the effect factors of cultivation on batch fermentation course in 5 L bioactor were fully discussed. Pilot scale was carried out in 70 L bioactor, elastase was purified by serials purification steps and its characteristics were determined. Complex cryoprotectant was screened and optimized by orthogonal experimental design. Storage period of elastase preparations was evaluated by accelerated storage test. Aqueous two- phase system was researched on elastase purification from broth. Ability of elastase hydrolysis on silk fibroin was investigated. The results were as follows:1) Batch cultivation process was studied in 5 L bioactor. Based on characteristics of cultivation course and analysis to dynamic data, the optimum cultivation strategy was suggested. The elastase produced by Bacillus sp. EL31410 was affected by temperature significantly. Cultivation temperature of 37°C was more suitable to cell growth at earlier cultivation stage while of 30 °C was benefit to get higher elastase activity, when compared to the results of batch cultivation at four kinds of cultivation mode, the conclusion was drawn that cultivation at 30 °C was appropriate for elastase produce. pH is one of important factors which affected fermentation course. It was discussed that pH-stat batch cultivation in 5 L bioactor was controlled at constant pH 6.0, 6.5, 7.0, 7.5, respectively. Cell growth was better at fermentation broth pH controlling at stable pH 6.5 or 7.0 than at others. However, the elastase activity was improved when cultivation at single pH 7.0. Analysis to the cultivation courses and dynamic data, two-stage pH- controlled strategy was adopted that broth pH wascontrolled at constant 6.5 from the beginning of cultivation to 24 h and was shifted to constant 7.0 form 24 h to the end. The two-stage pH strategy was carried out and the result was satisfied that the optimum elastase activity was achieved 699 U/mL when cultivated 48 h. When stirred speed was increased from 300 rpm to 400 rpm and the other cultivation condition was operated at 30 °C with two-stage pH controlled strategy in 5 L fermentor, cell grew well and elastase produced well, too. And the cultivation time to reach maximal elastaseactivity advanced 6 h.2) Pilot test was carried out in 70 L bioactor. Cultivation parameters were adjustedaccording to those in 5 L except temperature and pH. After cultivation 33 h, the maximal elastase activity was 645 U/mL which close to that of in 5 L fermentor. A simple model was proposed using Logistic equation for growth, the Luedeking-Piret equation for elastase production and Luedeking-Piret-like equation for glucose consumption. The models were as follows:3) The broth collected after cultivation for hours was centrifuged at 3000 rpm and the upper was salted out using (NH4)2SO4. Purified elastase was obtained after the two steps chromatography purification with DEAE Saphadex A 50 and Saphadex G 100 later. One clear band was shown by SDS-PAGE and the relative molecular weight was 31820 Da. Some enzymatic characteristics of purified elastase were evaluated: the optimum temperature for elastase hydrolysis action on elastin was 50 °C and it was very stable at 30-50 °C. The optimum pH for elastase hydrolysis action on elastin was pH 7.4 and pH 8.2 in boric acid buffer and in Tris-HCl buffer, respectively. Elastase behaved stable at pH 7-8. Some ions in low concentration such as Ca2+ and K+ showed active action on elastase while others such as inactive elastase activity to some degree. Elastase catalyzed hydrolysis action on solubleelastin more str更多还原